Abstract

Studies of C. elegans vulval development have illuminated mechanisms underlying cell fate specification and elucidated intercellular signaling pathways [1]. The vulval precursor cells (VPCs) are spatially patterned during the L3 stage by the EGFR-Ras-MAPK-mediated inductive signal and the LIN-12/Notch-mediated lateral signal. The pattern is both precise and robust [2] because of crosstalk between these pathways [3]. Signaling is also regulated temporally, because constitutive activation of the spatial patterning pathways does not alter the timing of VPC fate specification [4, 5]. The heterochronic genes, including the microRNA lin-4 and its target lin-14, constitute a temporal control mechanism used in different contexts [6-8]. We find that lin-4 specifically controls the activity of LIN-12/Notch through lin-14, but not other known targets, and that persistent lin-14 blocks LIN-12 activity without interfering with the key events of LIN-12/Notch signal transduction. In the L2 stage, there is sufficient lin-14 activity to inhibit constitutive lin-12. Our results suggest that lin-4 and lin-14 contribute to spatial patterning through temporal gating of LIN-12. We propose that in the L2 stage, lin-14 sets a high threshold for LIN-12 activation to help prevent premature activation of LIN-12 by ligands expressed in other cells in the vicinity, thereby contributing to the precision and robustness of VPC fate patterning.

The 1° fate is specified normally, but the 2° fate is not, in lin-4(0) mutants

Scoring of markers and complete genotypes of strains shown in these figures are given in .(A) P6.p adopts the 1° fate in lin-4(0) mutants. "lin-4 gonad ablated": the L1 gonad was ablated in lin-4(0) mutants.(B) All VPCs respond to EGFR-Ras-MAPK signaling in lin-4(0). The 1° fate marker used was ayIs4[egl-17p::gfp]. Note also that the penetrance of 1° marker expression increases in P5.p and P7.p, consistent with loss of lateral signaling.(C) Direct transcriptional targets of LIN-12, which mark the 2° fate, are not expressed in lin-4(0) mutants. Another 2° fate marker, nIs106[lin-11p::gfp], is also not expressed in lin-4(0) ([]; see ).

lin-4(0) blocks the effects of constitutive LIN-12 activity, but not cleavage or nuclear access of the LIN-12 intracellular domain

(A) lin-4(0) suppresses the ectopic expression of the 2° fate marker nIs106[lin-11p::gfp] caused by constitutively active LIN-12. In addition, the Multivulva phenotype caused by lin-12(n137) and LIN-12(intraΔP) expressed in VPCs was completely suppressed by lin-4(0). For arEx1080, 100% (99/99) of lin-4(+) hermaphrodites had at least one ectopic pseudovulva but 0% (0/86) of lin-4(0) had a pseudovulva; for arEx1094, 99% (87/88) of lin-4(+) hermaphrodites but 0% (0/35) of lin-4(0) hermaphrodites had a pseudovulva.(B) Nuclear accumulation of the GFP-tagged LIN-12 intracellular domain appears to be unaffected in lin-4(0) mutants.

(A) High lin-14 activity near the time of lateral signaling blocks 2° fate specification. lin-4(0); lin-14(n355n679ts) has a highly penetrant lateral signaling defect at 15° that is relieved at 25°. Reducing lin-14 activity up to the L2 molt permits lateral signaling. See for details. ***, p < 0.001; N. S., not significant (Fisher's exact test).(B) Reduction of lin-14 activity in the L2 stage removes the normal block to constitutive lin-12 activity. A comparable level of arIs107[mir-61p::yfp] expression in the L3-stage VPCs in lin-12(n137) mutants was obtained in the L2-stage VPCs when lin-14 was reduced by growth at 25° during the L2 stage. **, p < 0.01; N. S., not significant (Fisher's exact test). We note that arIs107[mir-61p::yfp] was chosen for this experiment because it is specifically expressed in response to the lateral signal in P5.p and P7.p at the L3 stage; other markers display more dynamic patterns until the Pn.px stage and hence could not be used here.(C) Anatomical configuration of lag-2-expressing cells ([]; X. Zhang, X. Karp and I. G., unpublished observations) with respect to the VPCs in the L2 and L3 stages.(D) Representation of the relationship between lin-4, lin-14 and lin-12. Left, lin-14 activity in the L2 stage may prevent premature activation of LIN-12 by ligands produced by other cells such as neurons in the local environment. When lin-14 activity is further lowered in the L3 stage, LIN-12 can activate its targets upon receiving the lateral signal produced by P6.p. Right, schematic representation of gene activity over time. lin-14 activity is represented by a dashed line because we do not know whether it decreases as a step function upon lin-4 expression or gradually.